RESUMEN
Ureaplasma respiratory tract colonization stimulates prolonged, dysregulated inflammation in the lungs of preterm infants, contributing to bronchopulmonary dysplasia (BPD) pathogenesis. Surfactant protein-A (SP-A), a lung collectin critical for bacterial clearance and regulating inflammation, is deficient in the preterm lung. To analyze the role of SP-A in modulating Ureaplasma-mediated lung inflammation, SP-A deficient (SP-A-/-) and WT mice were inoculated intratracheally with a mouse-adapted U. parvum isolate and indices of inflammation were sequentially assessed up to 28 d postinoculation. Compared with infected WT and noninfected controls, Ureaplasma-infected SP-A-/- mice exhibited an exaggerated inflammatory response evidenced by rapid influx of neutrophils and macrophages into the lung, and higher bronchoalveolar lavage TNF-alpha, mouse analogue of human growth-related protein alpha (KC), and monocyte chemotactic factor (MCP-1) concentrations. However, nitrite generation in response to Ureaplasma infection was blunted at 24 h and Ureaplasma clearance was delayed in SP-A-/- mice compared with WT mice. Coadministration of human SP-A with the Ureaplasma inoculum to SP-A-/- mice reduced the inflammatory response, but did not improve the bacterial clearance rate. SP-A deficiency may contribute to the prolonged inflammatory response in the Ureaplasma-infected preterm lung, but other factors may contribute to the impaired Ureaplasma clearance.
Asunto(s)
Inflamación , Pulmón , Neumonía/microbiología , Neumonía/fisiopatología , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Ureaplasma/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/microbiología , Citocinas/metabolismo , Humanos , Recién Nacido , Inflamación/microbiología , Inflamación/patología , Pulmón/citología , Pulmón/metabolismo , Pulmón/microbiología , Ratones , Ratones Noqueados , Óxido Nítrico/metabolismo , Neumonía/patología , Proteína A Asociada a Surfactante Pulmonar/administración & dosificación , Proteína A Asociada a Surfactante Pulmonar/genéticaRESUMEN
The mechanisms by which moderate hypothermia (32 degrees C for 12-72 h) affect human cellular function are unclear. We tested the hypothesis that it produces broad changes in mRNA expression in vitro. Acute monocytic leukemia (THP-1) cells were incubated under control conditions (37 degrees C) or moderate hypothermia (32 degrees C) for 24 h. RNA was extracted, and the hypothermic response was confirmed by examining the expression of the cold-induced RNA-binding protein (CIRBP) gene by RT-PCR. Gene expression analysis was performed on seven sets of paired samples with Affymetrix U133A chips using established statistical methods. Sequences were considered affected by cold if they showed statistically significant changes in expression and also met published post hoc filter criteria (changes in geometric mean expression of > or =2-fold and expression calls of "present" or "marginal" in at least half of the experiments). Changes in the expression of selected sequences were further confirmed by PCR. Sixty-seven sequences met the criteria for increased expression (including cold-inducible genes CIRBP and RNA binding motif 3), and 100 sequences showed decreased expression as a result of hypothermia. Functional categories affected by hypothermia included genes involved in immune responses; cell growth, proliferation, and differentiation; and metabolism and biosynthesis. Several heat shock proteins (HSPs) showed decreases in expression. Moderate hypothermia produces substantial changes in gene expression, in categories potentially of systemic importance. Cold exposure without rewarming decreased the expression of several HSPs. These in vitro findings suggest that prolonged hypothermia in vivo might be capable of producing physiologically relevant changes in gene expression by circulating leukocytes.
Asunto(s)
Regulación de la Expresión Génica , Hipotermia/metabolismo , Monocitos/metabolismo , Línea Celular Tumoral , Ciclofilina A/genética , Ciclofilina A/metabolismo , Bases de Datos Genéticas , Perfilación de la Expresión Génica/métodos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Hipotermia/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Hypothermia (HT) has been associated with both beneficial and detrimental consequences in various pathophysiological states. While HT is generally thought to have anti-inflammatory and cytoprotective effects, we have previously shown that moderate in vitro HT prolongs TNF-alpha production by LPS-stimulated mononuclear phagocytes, in part by prolonging TNF-alpha gene transcription and activation of the pleiotropic transcription factor NF-kappaB. In this study, we have further characterized the effect of moderate (32 degrees C) and marked (28 degrees C) HT in human monocytic THP-1 cells by showing that even short (2 h) exposure to HT followed by a return to normothermic conditions for 22 h resulted in augmented and prolonged production of TNF-alpha. Production of heat shock protein 72 and activation of heat shock factor 1 are not affected by HT in these studies, suggesting that the effect is not part of a generalized stress response. Using immunoblotting, we have shown that HT augments phosphorylation of IKK-beta and IKK-alpha (up to an 8-fold increase at 28 degrees C and a 3.6-fold increase at 32 degrees C vs. 37 degrees C). Furthermore, nuclear accumulation of NF-kappaB p65 was significantly prolonged in hypothermic cells (1.4- and 2.5-fold more nuclear p65 at 2 and 4 h at 28 vs. 37 degrees C). Reexpression of IkappaB-alpha, which contributes to the termination of NF-kappaB-dependent transcription, was delayed several hours in HT-exposed cells. Thus we have shown that clinically relevant HT alters both cytosolic and nuclear events responsible for NF-kappaB activation and deactivation. Enhanced NF-kappaB activation may contribute to the immunomodulatory effects of HT in various clinical settings.